Fate of Pharmaceuticals in the Environment and in Water Treatment Systems - Chapter 13 pot

Hormones in Waste from Concentrated Animal Feeding Operations 293animal manure need to be disposed from AFOs annually.28To ensure that the ers and managers of CAFOs take appropriate act

Concentrated Animal

Feeding Operations

Z Zhao, K.F Knowlton, and N.G Love

Contents

13.1 Introduction 292

13.2 Background 292

13.2.1 Concentrated Animal Feeding Operations (CAFOs) in the United States 292

13.2.2 Hormones and CAFO 293

13.3 Hormones in CAFOs 295

13.3.1 Natural Estrogens 295

13.3.2 Biosynthesis of Estrogens 296

13.3.3 Metabolism of Estrogens 297

13.3.4 Excretion of Estrogens 298

13.3.5 Degradation of Estrogens 300

13.4 Natural Androgens 302

13.4.1 Biosynthesis of Androgens 302

13.4.2 Metabolism of Androgens 302

13.4.3 Excretion of Androgens 303

13.4.4 Degradation of Androgens 304

13.5 Natural Progestagens 304

13.5.1 Biosynthesis of Progesterone 304

13.5.2 Metabolism of Progesterone 305

13.5.3 Excretion of Progesterone 306

13.5.4 Degradation of Progesterone 306

13.6 Hormone Growth Promoters 306

13.6.1 Naturally Occurring Hormonal Growth Promoters (HGPs) 307

13.6.2 Synthetic HGPs 307

13.7 Routes of Hormone Loss from CAFOs 310

13.7.1 Soil and Runoff 311

13.7.2 Streams and Rivers 312

13.7.3 Groundwater 312

13.8 Fate of Hormones during Manure Storage, Treatment, and Land Application 313

13.8.1 Conventional Manure Storage and Treatment Systems 313

13.8.1.1 Manure Handling on Dairy Farms 313

13.1 INTRODUCTION

Concentrated animal feeding operations (CAFOs) are the largest of the 238,000 live-stock farms or animal feeding operations (AFOs) in the United States.29The U.S Environmental Protection Agency (EPA) defined AFOs as livestock operations where animals are maintained or confined for more than 45 days in 1-year period,19and AFOs that meet certain animals threshold numbers and on-farm situations are catego-rized as CAFOs An estimated 15,500 AFOs (7% of the total) constitute CAFOs.29

Nitrogen (N) and phosphorus (P) pollution, pathogens, and odor of manure discharging from CAFOs have been a great concern for several decades.57,72,113In recent years, hormones (estrogens, androgens, progesterone, and various synthetic hormones) contained in the manure from CAFOs have generated wide interest because of their endocrine disrupting effects.48,79,90 When manure is land applied, part of these hormones may enter water systems through runoff or leaching24,37,63,102

and may cause developmental and reproductive impairment in aquatic animals The effective levels of these hormones may be as low as nanograms per liter of water.17A high incidence of intersexuality (feminization) was observed in a wide population of male roaches in the United Kingdom.64After exposure to cattle feedlot effluent, both testosterone synthesis and testis size decreased in male fathead minnows.105

This chapter focuses on the occurrence, persistence, treatment, and transforma-tion of natural and exogenous hormones in waste from CAFOs, including dairy, beef, poultry, swine, and horse farms The biochemistry, physiological functions, excretion, degradation, and environmental effects of these hormones are discussed

13.2 BACKGROUND

1 3.2.1 C ONCENTRATED A NIMAL F EEDING O PERATIONS (CAFO S )

IN THE U NITED S TATES

AFOs in the United States have been identified as one of the leading sources of impairment for all kinds of water bodies, because more than 500 million tons of

13.8.1.2 Poultry Farms 313

13.8.1.3 Swine Farms 314

13.8.1.4 Beef Cattle Operations 314

13.8.2 Innovative Manure Treatment Systems 315

13.8.3 Composting 315

13.8.4 Anaerobic Digestion 315

13.8.5 Nitrification and Denitrification 315

13.8.6 Chemical and Biological Phosphorus Removal 316

13.8.7 Aeration of Dairy Manure 316

13.9 Other Best Management Practices to Reduce Hormone Loss from CAFOs 317

13.9.1 Constructed Wetlands 318

13.9.2 Buffer Strips 319

13.9.3 Controlled Stream Access 319

13.10 Future Research Needs Regarding Manure Treatment 320

13.11 Conclusion 320

References 321

Hormones in Waste from Concentrated Animal Feeding Operations 293

animal manure need to be disposed from AFOs annually.28To ensure that the ers and managers of CAFOs take appropriate actions to manage manure effec-tively, CAFOs are subject to the National Pollution Discharge Elimination Systempermitting requirements and the Effluent and Limitations Guidelines and Standardsunder the Clean Water Act.29

own-CAFOs are divided into large and medium categories (Table13.1) Large CAFOsare defined only by animal numbers, while medium CAFOs are defined by both ani-mal numbers and on-farm conditions.29In addition to animal number requirement,medium CAFOs are also “discharging pollutants directly or indirectly via either aman-made ditch, flushing system, or other similar man-made devices into watersthat originate outside of and pass over, across, or through the facility or the animalsconfined in the operations have direct contact with water.”29Swine CAFOs are fur-ther divided by animal size, and poultry operations with both wet and dry manurehandling systems are included

The estimated numbers of CAFOs that need permits under the revised CAFOregulations announced by EPA in 2003 are listed inTable13.2.29Sixty percent ofthe CAFOs are hog and dairy farms CAFOs only account for a small percentage

of all livestock farms, but they contribute about 50% animal manure production(Table13.3).68

13.2.2 H ORMONES AND CAFO

Hormones are synthesized from specialized glands in the endocrine system andare excreted at very low quantities in urine and feces.86,95The hormones in animal

TABLE 13.1

Size Thresholds of CAFOs for Different Species of Livestock

Mature dairy cows 200–699 (milked or dry) >700 (milked or dry)

Chickens (other than laying hens) 3 37,500–124,999 >125,000

1 Cattle includes heifers, steers, bulls, and cow/calf pairs

2,6 If the AFO uses a liquid manure handling system

3,4,5 If the AFO uses other than a liquid manure handling system

Source: Adapted from U.S EPA [Reference 29].

manure that have important environmental effects include estrogens (estrone, diol, and estriol), androgens (testosterone), and progestagens (progesterone) If thesehormones enter the water system through runoff and leaching following manureland application, they may alter or disrupt the functions of the endocrine system andcause adverse effects to organisms.27The adverse effects may include mimicking orblocking receptor binding, or altering the rate of hormone synthesis or metabolismthrough interactions with the endocrine system.95

estra-It was predicted that about 1500 kg estrone and estradiol are excreted each year

by farm animals in the United Kingdom (Table13.4),66about four times more thanthe total estrogens from humans Forty-nine tons of estrogens, 4.4 tons of andro-gens, and 279 tons of gestagens were excreted by farm animals in the United States

in 2002,79and cattle production contributes about 90% of estrogens and gestagensand 40% of androgens These estimates are questionable as data available may not

TABLE 13.3

Manure Available for Land Application

Animals

Total Manure (Billion lbs)

By CAFOs (Billion lbs)

Manure from CAFO (% of total)

Numbers of CAFOs in the United States for

Different Types of Operations

Animals Medium CAFOs Large CAFOs

Source: Adapted from U.S EPA (Reference 29).

Hormones in Waste from Concentrated Animal Feeding Operations 295

be sufficient for accurate calculation of the total mass of estrogens excreted.48It isclear, however, that large amounts of hormones from CAFOs are released into theenvironment each year

Synthetic hormones considered to be endocrine disruptors are commonly used inCAFOs for different purposes Although banned in the European Union, hormonalgrowth promoters (HGPs) are widely used by the largest cattle-producing countries

in the world, including the United States, Australia, Argentina, and Canada.139Threesynthetic hormones, zearalanol, trenbolone acetate (TBA), and melengestrol acetate(MGA), have been licensed for animal production Also used are the natural hor-mones testosterone, 17C-estradiol, and progesterone.81

These HGPs are primarily used in the beef cattle industry, as exogenousandrogens and estrogens have little efficacy in pigs.14 In the dairy industry,progesterone-releasing implant devices are approved in lactating cows for estroussynchronization.114The use of these hormones in CAFOs has raised concerns withthe increased exposure of endocrine disruptors to the environment.126,160

13.3 HORMONES IN CAFOS

13.3.1 N ATURAL E STROGENS

Estrogens are hormones that are mainly responsible for the development of femalesex organs and the secondary sex characteristics (Figure 13.1) The naturally occur-ring estrogens in livestock include estrone or E1 (3C-Hydroxyestra-1,3,5(10)-trien-

TABLE 13.4

Predicted Total Excretion of Estrogens from the Human and

Farm Animal Populations in the United Kingdom (2004)

Type

Population (million)

Estrone (kg/year)

Estradiol (kg/year)

Discharge Percentage (%) 1

1 Based on the total amount of estrone and estradiol

2 The combined amount of estrone and estradiol is 260 kg/year

3 NC—not calculated for insufficient data

Source: Adapted from Johnson et al (Reference 66).

17-one), 17C-estradiol or E2 (estra-1,3,5(10)-triene-3,17C-diol), and estriol or E3(estra-1,3,5(10)-triene-3,16C,17C-triol) 17B-estradiol is the optical isomer of 17C-estradiol with the hydroxyl group at C-17 pointing downward from the molecule,and 17C-estradiol has the hydroxyl group pointing upward.48Natural estrogens areslightly soluble in water, moderately hydrophobic, and are weak acids with low vola-tility (Table13.5) 17C-estradiol is the most potent natural estrogen, and the relativeestrogenic potencies of estrone and estriol relative to 17C-estradiol (1.0) are 0.38 and2.4 × 10–3, respectively, based on the yeast estrogen screen assay.122

13.3.2 B IOSYNTHESIS OF E STROGENS

The major source of estrogens in nonpregnant females is the ovary (granulosa cells),and the placenta produces the majority of estrogens during pregnancy.54 Othersources may include the adrenal cortex, adipose tissue, muscle, kidney, liver, andhypothalamus The main pathways of biosynthesis of estrogens are shown inFig-ure13.2.38The synthesis starts with cholesterol, which is converted to pregnenolone,and subsequently 4-androstenedione and testosterone The two androgens are thenhydroxylated at C-19, and the resulted 19-hydroxyl groups are oxidized and furtherremoved The final products will be estrone and 17C-estradiol from androstenedi-one and testosterone, respectively 17C-estradiol can then be converted to estriol

OH

Estriol Estrone

Estradiol

FIGURE 13.1 Molecular structures of natural estrogens.

TABLE 13.5

Physicochemical Properties of Natural Estrogens

Property Estrone Estradiol Estriol

2 Kow—octanol-water partition coefficient

3 PKa—acid ionization constant

Source: Adapted from Hanselman et al (Reference 48).

Hormones in Waste from Concentrated Animal Feeding Operations 297

by 16C-hydroxylase The biosynthesis pathways of 17B-estradiol are not entirelyknown.148Generally it is synthesized from aromatization of epitestosterone by thecytochrome P450 aromatase.36

13.3.3 M ETABOLISM OF E STROGENS

After synthesis and secretion, estrogens go through a series of metabolic pathways inthe liver, kidney, gastrointestinal tract, and target tissues Estrone and 17C-estradiolare interconvertible by 17C-hydroxysteroid dehydrogenase.120All estrogens may beconverted to glucuronides or sulfates by UDP-glucuronosyltransferase and sulfo-transferase, and glucuronidase and sulfatase will hydrolyze estrogen conjugates back

to free forms, as shown inFigure13.3.115

These conjugated estrogens are not involved in estrogen receptor-mediatedactivity.166Estrogen sulfates have a much longer half-life and higher concentrations

in human circulation than the free forms,108 so sulfation and desulfation may be



FIGURE 13.2 Biosynthesis pathways of natural estrogens (Adapted from Fotherby.38 )

important in the regulation of biologically active or free estrogens in the body.52

Compared with estrogen sulfates, much less attention has been paid to estrogenglucuronides.166

Hepatic hydroxylation of estrogens at C-2 and C-4 forms catechol estrogens (CEs)with less hydroxylation at C-4 than at C-2 in humans and most mammals.103Thehydroxylated estrogens can form sulfates and glucuronides, or with the presence ofcatechol-O-methyltransferase, the CEs are methylated to form 2- or 4-O-methylethersand then excreted as shown inFigure13.4.115The CEs can also be oxidized furtherinto quinones or semiquinones with subsequent glutathione conjugation.9,124

In humans about half of the estrogen conjugates that enter or are formed in theliver will be excreted into the intestine through bile for enteroheptatic circulation(intestinal reabsorption and reentering the liver for metabolism) or excretion infeces.41Generally estrogen conjugates are hydrolyzed considerably in the intestine,and estradiol is converted to estrone to a large extent.1Reconjugation can occur inthe intestinal mucosal cells, and some of the conjugated and free estrogens are thenreabsorbed to the bloodstream and either reenter bile or are transported to the kidneyfor urinary excretion.2

13.3.4 E XCRETION OF E STROGENS

Estrogens are mainly excreted through urine and feces In feces, estrogens mainlyexist in free forms, while urinary estrogens are mostly conjugated.138 17C-estra-diol, 17ß-estradiol, and estrone (free and conjugated) account for more than 90% ofthe excreted estrogens in cattle,48but 17C-estradiol is rarely excreted by swine and

O O

Estradiol sulfate Estrone sulfate

Hormones in Waste from Concentrated Animal Feeding Operations 299

poultry In cattle 58% of the total estrogen excretion is via the feces,59while swineand poultry excrete 96% and 69% of estrogens in urine, respectively.3,106

Total estrogen excretion by cattle is clearly quantitatively significant, but tively few data are available 174 μg per day of total estrogens was excreted throughcow urine in days 6 to 25 of lactation.30In cattle, 11.6 ng/g estrone, 60.0 ng/g 17C-estradiol, and 33.6 ng/g 17ß-estradiol, respectively, were found in the feces 5 daysbefore parturition (giving birth), but no daily excretion rate data for estrogens wereavailable.53Total estrogen content (estradiol plus estrone) was reported to be 28 ng/g

rela-of dry broiler litter (“Litter” is the term used for the combination rela-of manure andwood shaving bedding material.137) Similarly, 30 ng/g of 17C-estradiol was found

in broiler chicken manure.10 Table13.6 shows the calculated total daily excretion

HO

2-OH estradiol

4-OH estradiol Estradiol-3,4-Quinone

Estradiol-2,3-Quinone HO

Methylation and Conjugation Glutathione Conjugation

FIGURE 13.4 Conjugation of catechol estrogens and estrogen quinines (Adapted from

Urinary Excretion Total Excretion

Source: Adapted from Lange et al (Reference 79).

of estrogens for cattle, pigs, and sheep.79Several factors, such as age, diet, season,health status, and diurnal variation may contribute to variation in excretion rates.130

13.3.5 D EGRADATION OF E STROGENS

Degradation of estrogens is a complicated process and may include deconjugation,dissipation, and mineralization.61,83,145A rapid biodegradation of 17C-estradiol andits related metabolites were reported by sewage bacteria under both aerobic andanaerobic conditions.82The same kind of result occurred during aerobic batch incu-bation with activated sludge from a sewage treatment plant.145

The hydrolysis or cleavage of sulfate or glucuronide of conjugated estrogens iscalled deconjugation It was suggested that some natural fecal bacterial and enzymesmay degrade estrogen metabolites several hours after sampling if no preservative

is added or samples are not put into cold storage.70The fecal microorganism erichia coli (ubiquitous in the digestive tract) is capable of producing large quanti-

Esch-ties ofC-glucuronidase.20,60and has been considered to be responsible for estrogen

glucuronides deconjugation to some extent Escherichia coli has a weak arysulfatase

activity,131so it is possible that portions of estrogen sulfates are left intact This mayexplain why several studies reported estrogen sulfates in sewers,145sewage treatmentplants,20and river water.121No estrogen glucuronides were detected in sow feces, andafter incubation of estrone conjugates at 20°C for 30 min in fecal suspension, 90% ofestrone glucuronide was deconjugated, but estrone sulfate was not hydrolyzed.156

The dissipation of estrogens refers to the decrease in extractable concentrations,and the possible dissipation pathways include conversion of estradiol to estrone andsubsequent formation of nonextractable residues.18Mineralization is the final deg-radation of estrogens to CO2, water, and other compounds through cleavage of thephenolic ring The degradation pathways for dissipation and mineralization have notbeen clearly understood, but both biotic and abiotic pathways are possible for estrogendegradation as shown inFigure13.5.71Under aerobic conditions, the introduction ofhydroxyl groups by mono- and dioxygenase ring cleavage, and final decarboxylation,are the key steps for degradation of phenolic compounds.128Anaerobes may degradephenolic compounds through hydroxylation and carboxylation followed byC-oxida-tion to CO2.11With the assistance of TiO2, estrogens are degraded chemically at thephenol ring first and then DEO (10F-17C-dihydroxy-1,4-estradien-3-one)
is produced

as an intermediate for further degradation.104Because of the similarity in their basicstructures, the degradation of androgens and progesterone is expected to go throughthe same pathways in terms of the final mineralization

Photodegradation has also been suggested as another mechanism of estrogen sipation and mineralization.35,97Most reported work focuses on estrogens in manure-amended soil and biosolids from wastewater treatment systems; only a few studieshave explored degradation of estrogens in stored manure Estrogen concentrationsdecreased significantly in broiler litter at pH 5 and 7 after 1 week incubation.137Thetotal estrogens were reduced by 80% in cattle feces following 12 weeks of incuba-tion at 20 to 23°C.129Substantial losses of 17C-estradiol (90%) and total estrogens(40%) occurred without acidification and cold storage in press-cake samples of dairy

dis-manure.116

Hormones in Waste from Concentrated Animal Feeding Operations 01

HO

O OH

Tricarboxylic Acid Cycle OH

OH

OH HO

COOH (a)

Ring Cleavage

Tricarboxylic Acid Cycle HO

13.4 NATURAL ANDROGENS

Natural androgens are C-19 steroids that possess androgenic activities (stimulatingand maintaining masculine characteristics), including testosterone, 5B-dihydrotes-tosterone, 5B-androstane-3C,17C-diol, and three weakly androgenic steroids—4-androstenedione, dehyroepiandrosterone (DHA), and androsterone—as shown in

Figure13.6.43 Testosterone and androsterone are less soluble in water and morehydrophobic compared to estrogens as shown in Table13.7.86 The 17C-hydroxylgroup accounts for most of the androgenic activities, and oxidation of the 17C-hydroxyl group to a 17-oxo group will cause an 80% loss of the androgenic potency.42

Epitestosterone testosterone) is a natural optical isomer of testosterone testosterone), but its biological activity has not been fully clarified.142 Androgensare important for the development of male sex organs and the maintenance of thesecondary sex characteristics.8

(17C-13.4.1 B IOSYNTHESIS OF A NDROGENS

Androgens are largely synthesized in the testes, secondly in the adrenal cortex, and to

a limited extent in the ovaries and placenta As shown inFigure 13.2, pregnenolone isalso the precursor for androgen biosynthesis, and it goes through hydroxylation at C-17and subsequent removal of the side-chain.42Then the resulted DHA is metabolized intotestosterone through either 5B-androstane-3C,17C-diol, or 4-androstenedione Anotherpathway starts with progesterone converted from pregnenolone, and then hydroxyl-ation at C-17 and removal of the side-chain gives 4-androstenedione Finally, testoster-one is formed from 4-androstenedione by 17C-hydroxysteroid dehydrogenase

Hormones in Waste from Concentrated Animal Feeding Operations 303

testosterone is converted to 5B- and/or 5C-dihydrotestosterone (DHT) as shown inure13.7.1435a-DHT binds to human androgen receptor with twofold higher affinity thantestosterone, while testosterone dissociates from the receptor fivefold faster than 5B-DHT.45The primary pathway to inactivate testosterone and DHT is the oxidation of the17-hydroxy group resulting in androstanedione and androstenedione.42Furthermore,the reduction of the keto group at C-3 produces four metabolites (epi)androstanedioland (epi)androsterone As with estrogens, testosterone can also be conjugated to gluc-uronic acid or sulfate directly or following hydroxylation.159,161

Fig-13.4.3 E XCRETION OF A NDROGENS

Similar to estrogen metabolites, the metabolites of androgens formed in the liverhave three destinies: returning back to the circulation, excretion in the urine, orexcretion through bile for enterohepatic circulation and fecal excretion In cattleurine, the main metabolites of testosterone are three isomers of androstane-3,17-

Physicochemical Properties of Natural Androgens

Property Testosterone Androsterone

diol, 5C-androstan-3B-ol-17-one, and epietiocholanolone.123In the intestine, gen metabolites are subjected to hydrolase, dehydroxylase, reductase, and epimeraseactivities of the bacteria.123

andro-Little data are available about androgens in livestock urine and feces; 133 μgand 250 μg testosterone were reported in per kg of broiler litter (both sexes) andbreeder litter,137but only 30 μg/kg and 20 to 30 μg/kg of testosterone equivalentswere detected in broiler litter (both sexes) and breeder layers, respectively.90Thesedifferent observations may reflect the differences associated with chickens (breed,age) and manure treatment The androgenic activity in manure from pregnant dairycows was determined to be 1737 ng testosterone equivalents/g dry weight.90Theestimated yearly total excretion of androgens is 120 mg, 390 mg, 670 mg, and 3.4 mgfor male calves, bulls, boars, and laying hens, respectively.79Based on these excre-tion data, about 4.4 Mg of androgens were excreted to the environment from farmanimals in the United States in 2000.79

13.4.4 D EGRADATION OF A NDROGENS

As with estrogens, testosterone may be degraded into extractable or nonextractableproducts, or completely mineralized into carbon dioxide.40The degradation processdepends on the organic matter, moisture, temperature, and oxygen availability of thematrix After 23 weeks incubation in poultry litter with water potential –24 MPa, anaverage of less than 2, 11, and 27% of the radiolabelled testosterone was mineral-ized to14CO2at 45, 35, and 25°C, respectively.51Aerobic composting for 139 daysdecreased the average concentration of testosterone in poultry manure from 115 ng/g

to 11 ng/g (dry basis), which means a 90% reduction in potent hormones.47

Microbial activity plays an important role in testosterone degradation Jacobsen

et al.61 observed rapid conversion of testosterone to 4-androstene-3,17-dione withinthe mix of soil and nonsterilized swine manure, and this effect was totally absent insoil amended with sterilized manure Mineralization of14C-testosterone decreased inmanured soil, and did not occur in sterilized soil.61Comamonas testosterone, a gram-

negative bacterium, can metabolize testosterone as its sole carbon and energy source.56

The possible mechanisms include dehydrogenation of the 17C-hydroxyl group, ration of the A-ring, hydroxylation, and final metacleavage of the bone structure

desatu-13.5 NATURAL PROGESTAGENS

Progestagens (also called progestogens or gestagens) are hormones that produceeffects similar to progesterone, the only natural progestagen All other progestagensare synthetic and are often referred to as progestins

13.5.1 B IOSYNTHESIS OF P ROGESTERONE

The two main sources of progesterone in female livestock are the corpus luteum (or

“yellow body,” a gland that forms on the surface of the ovary following ovulation)and the placenta During the estrous cycle, the ovarian corpus luteum producesmore progesterone as it matures If pregnancy does not occur, prostaglandin F2Bsecreted by the uterus will cause corpus luteum to regress, triggering a decline

Hormones in Waste from Concentrated Animal Feeding Operations 305

in progesterone production During gestation, the placenta is the dominant source

of progesterone to maintain normal pregnancy As shown inFigure13.2 andure13.8,42progesterone is also synthesized from cholesterol via pregnenolone Firstcholesterol is hydroxylated at C-20 and C-22 to form the subsequent intermediate20,22-dihydroxycholesterol, and then pregnenolone and isocaproic aldehyde areformed through side-chain cleavage Finally, pregnenolone is converted to proges-terone by hydroxysteroid dehydrogenase The physiochemical properties of preg-nenolone and progesterone are shown inTable13.8.26,87

FIGURE 13.8 Biosynthesis pathways of progesterone from cholesterol.

also determined, and more than 55% of the conjugated metabolites was 3B,20B-diol glucuronide 3C-hydroxy-5B-pregnan-20-one 3 sulfate was reported to bethe major metabolite in pregnant sheep plasma after injection of14C-progesterone.150

5C-pregnane-Progesterone also serves as a precursor for estrogen and testosterone synthesis,

as shown inFigure 13.2 Hydroxylation on progesterone may happen in the adrenalgland, leading to cortisol and aldosterone production.76

13.5.3 E XCRETION OF P ROGESTERONE

Progesterone in the urine and feces has been used to monitor the estrous and nancy status of livestock.58,80,99 Two pregnenolones and two pregnanediols weredetected in pregnant sow urine.25 Significant positive correlations were foundbetween fecal and plasma progesterone concentrations during the ovarian cycle in

preg-beef cattle (r = 0.7), and the fecal progesterone ranged between 9 and 139 ng/g,

depending on the cycle phases.58An even higher correlation (r = 0.98) was found

between plasma progesterone and fecal gestagens (progesterone and its metabolites)

in sows, and the average concentration of fecal gestagens was 71 to 497 ng/g duringthe ovarian cycle.99Fifty percent of the radiolabeled progesterone was recovered inbile/feces in 1 to 2 days following intravenous injection, and only 3% of the dose wasfound in urine.31

13.5.4 D EGRADATION OF P ROGESTERONE

Data regarding progesterone degradation in the environment are scarce Plourde

et al.110 reported the biotransformation of progesterone by spores and vegetative

cells of microorganisms found in soil Some fungi contained in Rhizopus nigricans

transformed progesterone into a mixture of 11B-hydroxy-4-androstene-3,17-dioneand 11B-hydroxy-1,4-androstadiene-3,17-dione.111The side-chain progesterone was

degraded by some species of Aspergillus flavus; 4-androstene-3,17-dione,

testoster-one, and testololactone were the main degraded metabolites.100Progesterone wasalso detected up to 6 ng/g in river sediments in some Spanish rivers.88

13.6 HORMONE GROWTH PROMOTERS

In both humans and animals, sex steroids such as estrogens, testosterone, and gesterone regulate growth and development This has led to the use of hormonalgrowth promoters (HGPs), natural sex steroids or their synthetic counterparts, inmeat animals to increase feed efficiency and weight gain The first approved syn-thetic estrogen was diethylstilbestrol (DES) in 1954 Because of its carcinogenicpotential, DES was banned for all the use in cattle production by the U.S Food andDrug Administration (FDA) in 1979.49

pro-Currently, six different hormones are approved for such use in the United States,including three natural hormones (17C-estradiol, testosterone, and progesterone),and three synthetic compounds that mimic the functions of these hormones (trenbo-lone acetate, zeranol, and melengestrol acetate) The chronology of the use of thesehormones in cattle in the United States is listed inTable13.9.118

Hormones in Waste from Concentrated Animal Feeding Operations 307

13.6.1 N ATURALLY O CCURRING H ORMONAL G ROWTH P ROMOTERS (HGP S )

The direct effects of exogenous 17C-estradiol administration on farm animals (calves,heifers, steers, lambs) include enhanced protein deposition in skeletal muscle andreduced nitrogen excretion94; growth performance is increased by 5 to 15% Testos-terone or other androgens are less active as compared to estrogens in cattle or lambs,probably because there are fewer androgen receptors than estrogen receptors.125

The anabolic mode of action of steroidal hormones has been well established

in the past two decades (Figure 13.9) The anabolic effects are mediated directly byseveral organs and tissues (liver, bone, skin, and other tissues) and indirectly via thesomatotropic axis involving growth hormone and insulin-like growth factor I

13.6.2 S YNTHETIC HGP S

The structures of TBA (17C-acetoxyestra-4,9,11-triene-3-one), MGA 6-methyl-16-methylene-pregna-4,6-diene-3,20-dione), and zeranol are shown inFig-ure 13.10 The physiochemical properties of the three synthetic hormones are shown

(17C-acetoxy-inTable13.10.32–34Compared to estrogens, TBA is more soluble in water, and MGAand zeranol have a much higher vapor pressure

TBA mimics the activity of testosterone and is administered as a subcutaneousimplant either alone or coupled with 17C-estradiol The anabolic effect of TBA isbased on its androgenic and antiglucocorticoid activity.93In heifers the major path-way of TBA metabolism is formation of 17B- and 17C-trenbolone (TBOH) throughhydroxylation on C-17, and subsequent oxidation and reduction on the formedhydroxyl.112TBA has 8 to 10 times greater anabolic effects than testosterone,126but

TABLE 13.9

Chronology of the Use of Anabolic Agents in the U.S Cattle Industry

1956 Estradiol benzoate/progesterone implants approved for steers

1958 Estradiol benzoate/testosterone propionate implants approved for beef heifers

1968 Oral melengestrol acetate approved for beef heifers

1969 Zearnol implants (36 mg) approved for cattle

1982 Silastic estradiol implant approved for cattle

1984 Estradiol benzoate/progesterone implants approved for beef calves

1987 Trenbolone acetate implants approved for cattle

1991 Estradiol/trenbolone acetate implants approved for steers

1993 Bovine somatotropin approved for use in lactating cows

1994 Estradiol/trenbolone acetate implants approved for use in heifers

1995 72-mg zeranol implants approved for beef cattle

1996 Estradiol/trenbolone acetate implants approved for stocker cattle

Source: From Raun and Preston (Reference 118).

TABLE 13.10

Physiochemical Properties of Synthetic Hormones

FIGURE 13.10 Chemical structures of TBA, MGA, and Zeranol.

FIGURE 13.9 Anabolic modes of action of steroid hormones (Adapted from Meyer.94 ) GHRH = Growth hormone releasing hormone; GH = Growth hormone; IGF-I = Insulin-like growth factor-I.

Hypothalamus

Pituitary GHRH

GH Fat Liver Bone

Muscle

IGF-I Skin Digestive Tract Epithelium

Kidney Other

Tissues

Somatostatin

Estrogens Androgens

Hormones in Waste from Concentrated Animal Feeding Operations 309

17B-TBOH has only 2% of the androgenic potency, 5% of the anabolic effect,112and 4%the affinity to the recombinant human androgen receptor compared to that of TBA.7

As with the natural steroidal hormones, little data are available on the excretion

or fate of synthetic HGPs in manure storage 17B-TBOH and 17C-TBOH ranged from

10 to 120 ng/L and 10 to 20 ng/L in the liquid discharge from a beef feedlot.23Thehalf-lives of the two metabolites were about 260 days in liquid cattle manure.126

Zeranol, also called C-zearalanol, is a resorcylic acid lactone The relatedcompounds of zeranol are shown inFigure13.11, including zearalanone, taleranol(C-zearalanol), C-zearalenol, zearalenon (mycotoxin), and C-zearalenol.140 Thesemetabolites can all be metabolized or converted into all the other compounds withvarying efficiencies.96,147Zeranol mimics the action of 17 C-estradiol and is oftenimplanted alone It is about equally potent to 17C-estradiol in inducing expression ofendogenous estrogen-regulated genes in human breast cancer MCF-7 cells.84

Zeranol and its metabolites do appear in manure, with concentrations est immediately after implantation C-zearalanol, zeranol, and zearalanone wereall detected in the urine of zeranol-implanted male veal calves, and C-zearalanolwas the major metabolite after 3 days following implantation, while zeranol was

high-a minor component during the whole excretion period (14 dhigh-ays).62This is in trast to the observation that zearalanone was the major metabolite in adult cattle.6

con-In steers treated with 36 mg zeranol, the concentrations of zeranol in urine andfeces were 13 ng/ml (peaked at day 8) for urine and from 10 to 15 ng/g (peaked atday 20) in feces.22 Zeranol concentrations in manure declined steadily during the

OH

O O

Zearalenon

OH

O O

O OH

Zearalanone

β-zearalenol O

OH

β-zearalenol O

O OH

β-zearalanol

Figure 13.11 Zeranol and its related metabolites (Adapted from Songsermsakul

et al.140)

The structures of TBA (17C-acetoxyestra-4,9,11-triene-3-one), MGA 6-methyl-16-methylene-pregna-4,6-diene-3,20-dione), and zeranol are shown inFig-ure 13. 10 The physiochemical properties of the. .. 11B-hydroxy-4-androstene-3,17-dioneand 11B-hydroxy-1,4-androstadiene-3,17-dione.111The side-chain progesterone was

degraded by some species of Aspergillus flavus; 4-androstene-3,17-dione,... either 5B-androstane-3C,17C-diol, or 4-androstenedione Anotherpathway starts with progesterone converted from pregnenolone, and then hydroxyl-ation at C-17 and removal of the side-chain gives 4-androstenedione